Human telomeres are complex nucleoprotein structures that maintain and monitor chromosome ends to coordinate genome integrity with cellular replicative capacity. Telomere maintenance aberrations are hallmarks of human cancer, as well as the source of several inherited syndromes associated with genetic instability and stem cell failure. Telomere repeat length and telomere repeat binding factors have well- established roles in genome maintenance and signaling. On the other hand, telomeric chromatin, which extends far beyond the terminal repeats, is also required for chromosome end-protection and signaling, but the precise structures and mechanisms of human telomeric chromatin are not well understood. Here, we propose to investigate the structure and function of human telomeric chromatin in normal and pathogenic conditions, including inherited telomere maintenance disorders and cancers. We will leverage newly developed methods for mapping and analyzing genome-wide sequence data to the human subtelomeric repeats and duplicons that comprise the regions adjacent to telomere repeats. Our published and preliminary data reveal that subtelomeric chromatin regulatory factors CTCF and cohesin regulate RNA polymerase II recruitment and the expression of telomere repeat containing RNA (TERRA). We have also found that DNA damage sensing factors, including p53, bind directly within subtelomeric sequence to promote TERRA transcription in response to DNA damage and to enhance telomere DNA protection and repair. We will use functional genomics methods to build a comprehensive epigenomic map of RNA transcripts, histone modifications, DNA modification, and chromatin regulatory factor interactions with human subtelomeric DNA. We have also initiated a new collaboration to identify novel and complex histone modification patterns specifically enriched at human telomeres. We will use CRISPR genome engineering methods to investigate the functional properties of CTCF and p53 binding sites in human subtelomeres. Finally, we will determine if telomere epigenomic patterns and chromatin structures are significantly altered in human cancers and inherited disorders associated with telomere dysfunction, and how these changes may alter telomere metabolism. These studies will provide important new information on the chromatin organization and epigenetic programming of human telomeres in normal development and disease.